1. Field of the Invention
The Invention is in the field of opthalmology, and concerns the use of a fundus camera to take autoflourescence images using high performance thin film optical interference filters.
2. Background of the Related Art
The diagnosis and treatment of retinal diseases is highly dependent on the ability to image the ocular fundus. Monochromatic and color photography provide means to record the fundus photographically. Fluorescein angiography affords a way to investigate and document vascular anatomy and physiology of the eye. In this test fluorescein dye is injected into the circulation, (usually in the arm) and the passage of the dye is photographed as it traverses the ocular circulation. The fluorescein dye passes through the vessels in the eye and can be recorded photographically.
FIG. 1 shows illustrative spectra for purposes of the following discussion, including lipofuscin, fluorescein and indocyanine green excitation and emission spectra, as well as the excitation and barrier filters for autofluorescence detection as employed by commercial scanning laser opthalmoscope systems. The lens autofluorescence shown is for an excitation of 485 nm, for comparison purposes. To image the fluorescein dye two optical filters are used. One filter, called an excitation filter, uses wavelengths generally centered between 490-500 nm to excite fluorescein. As shown in spectral curve 101, fluorescein can absorb, and potentially produce fluorescence from, wavelengths up to 530 nm. The other filter, called a barrier filter, is a low-pass filter having an upper cutoff at about 500 nm. Fluorescein fluoresces at a peak between 520-530 nm as illustrated by the spectral curve 102. Indocyanine green angiography (for which the applicable excitation (103) and emission (104) spectra are also shown in FIG. 1) is done in a somewhat similar fashion in that the dye is injected, and the fluorescence of the dye, which occurs in the near infra-red region (peaking at 825 to 835 nm), is photographed. With these dyes we could directly visualize the vessels of the ocular fundus and could indirectly obtain information about other layers of the fundus.
Another approach to imaging the fundus would be to cause naturally occurring fluorophores in the fundus to fluoresce and then record that fluorescence. There is a waste material that accumulates within cells, namely lipofuscin, that is highly autofluorescent.1-5 (All bibliographic references are listed by number in Table I.) Lipofuscin accumulates in cells as a normal consequence of life. Increased amounts of lipofuscin can accumulate through a variety of mechanisms. In many cell types, oxidative damage can increase the amount of lipofuscin present.6,7 Lipofuscin accumulation can occur in a number of different inherited diseases. In the eye, lipofuscin accumulates through a novel mechanism within a cell monolayer called the retinal pigment epithelium (RPE). The retina is the light sensitive structure within the eye. On the bottom surface of the retina there are photoreceptors, which are cells that have numerous small flat discs called outer segments. These outer segments are composed of retinoids used in the actual detection of light, proteins, and polyunsaturated fatty acids. Normal retinal function leads to the accumulation of autofluorescent fluorophores. These molecular components are capable of being damaged by light and oxygen. The photoreceptors shed the outer segments and the shed outer segments are phagocytized by the retinal pigment epithelium. The undamaged fatty acids and retinoids are removed from the outer segments and are recycled by the retinal pigment epithelium. However, a portion of the material within the outer segments is damaged. Some of this material proves to be very difficult for the retinal pigment epithelial cell to digest. This material is segregated by the cell into structures known as liposomes which contain the material lipofuscin. One important component of lipofuscin in retinal pigment epithelial cells is a molecule called A2E, which is formed from two molecules of trans-retinal and one molecule of phosphatidlyethanolamine.8 The composition of lipofuscin in retinal pigment epithelial cells is somewhat different than lipofuscin found in other cells of the body because of the unique job of the retinal pigment epithelium in processing the photoreceptor outer segments. Components of lipofuscin inhibit lysosomal protein degradation,4 are photoreactive,5 are capable of producing a variety of reactive oxygen species and other radicals,5 are amphiphilic, may induce apoptosis of the RPE,9 and mediates blue light induced RPE apoptosis.10 
There are sources of autofluorescence (i.e., fluorophores) within the eye other than lipofuscin. Precursors of A2E, such as A2PE-H2, A2PE, and A2-rhodopsin, all of which are autofluorescent, form in outer segments prior to phagocytosis by the RPE,11,12 There is not a significant accumulation of the autofluorescent material within the retina unless there is some disease process that limits the retinal pigment epithelium's ability to phagocytize the outer segments.13,14 The fluorescence spectra for the precursors of A2E peaks at wavelengths longer than A2E. The accumulation of autofluorescence within the ocular fundus occurs from fairly predictable mechanisms. By imaging the autofluorescence we can obtain not only anatomic information from the lipofuscin that is in retinal pigment epithelial cells, but we can make inferences about functional aspects of these retinal pigment epithelial cells as well.
Imaging Autofluorescence
Lipofuscin can be made to fluoresce and has a broad emission band (105 in FIG. 1) ranging from about 500 to beyond 750 nm.1 In opthalmology this intrinsic fluorescence is called autofluorescence to differentiate this process from that seen from administered dyes like fluorescein or indocyanine green. The intensity of fundus autofluorescence parallels the amount and distribution of lipofuscin.1,15,16 
If we shine excitation light into the eye to excite the lipofuscin, the resultant fluorescence can be detected. The major problem with such an approach is that there are structures in the eye in front of the retina that also fluoresce. The main culprit in this regard is the crystalline lens, which has a broad band of fluorescence (106 in FIG. 1) secondary to principally blue, but also green fluorophores especially as it develops nuclear sclerosis with age. The fluorescent emission from the crystalline lens varies with the excitation light used, the age of the patient, the amount of nuclear sclerosis, and the concurrent diseases that may be present such as diabetes. The lens fluorescence has a broad peak ranging from 500 to about 550 nm for the commonly used wavelengths for autofluorescence photography of the fundus. Therefore autofluorescence of the crystalline lens overlaps the fluorescence produced by fluorescein. Crystalline lens autofluorescence causes fluorescein angiograms to look washed out when taken of an eye with nuclear sclerosis—the autofluorescence of the lens adds to the fluorescence coming from the fundus to produce an image with low contrast. To produce useful autofluorescence images we need to be able to either reject or bypass the fluorescence of the lens.
Scanning laser opthalmoscopes have a confocal capability where only conjugate points on the fundus are imaged. Points not lying on the conjugate planes are rejected. So the autofluorescence of the lens can be rejected by a scanning laser opthalmoscope. This allows confocal scanning laser opthalmoscopes to use excitation 201 and barrier 202 filters (as represented in FIG. 2) similar to that used in fluorescein angiography to obtain autofluorescence photographs. The gain is turned up and usually a number of images are taken and then averaged to obtain a final image with less noise. However, autofluorescent images cannot be taken if a patient has previously received fluorescein dye. Increasing attenuation of the excitation light occurs with increasing nuclear sclerosis. The photographs obtained with a scanning laser are quite noisy and it is customary to average several images together to reduce the noise present. The software routines that perform this function commonly normalize the image, changing the grayscale values in the process. This makes objective measurement of grayscale values with the commercially available scanning laser impossible. The wavelengths commonly used for autofluorescence determination by the commercially available scanning laser opthalmoscope are absorbed by the macular pigment, which limits its ability to accurately image the central macula.
Delori, et al. used a 550 nm excitation filter with a glass absorbing filter centered at 590 nm. The camera system used a CCD camera cooled to −20 degrees C. and had a restricted field of view of 13 degrees “ . . . to minimize the loss in contrast caused by light scattering and fluorescence from the crystalline lens.” This system was capable of imaging autofluorescence, but the published images had low contrast and a 13 degree field of view is not acceptable for clinical practice.17 
The details of a fundus camera system that I previously developed for photographing autofluorescence in the retina are published.18 The applicable spectral characteristics are illustrated in FIG. 3, including the corresponding curves 301, 302 for the excitation and barrier filters, respectively, of that publication. This camera system used a bandpass filter centered at about 580 nm (yellow-orange) for excitation and a bandpass filter centered at about 695 nm (near infrared that extended into the infrared wavelengths) as a barrier filter. The wavelengths used are not expected to show much attenuation from nuclear sclerosis. Since the lens fluorescence occurs with wavelengths shorter than the upper cut-off of the barrier filter, lens autofluorescence is usually not much of a problem, unless the patient has severe degrees of nuclear sclerosis. The limitations of this system for autofluorescence include all of those a typical fundus camera would face, particularly for patients with small pupils. The absorption of fluorescein extends to at least 530 nm and thus the published filter stimulates fluorescein. The excitation filter used a metal dielectric coating to block the far infrared portions of the spectrum, which caused a reduction in the transmission of the desired wavelengths by about one-half. The barrier filter was placed into the near infrared region, which has some disadvantages. The first is that this range of wavelengths is on the far, declining, edge of the fluorescence spectrum of lipofuscin. The second is that the optical performance of the camera and the eye is better adapted to visible wavelengths. The third disadvantage of this filter system was that the far infrared portion of the returning light was not blocked by the barrier filter. Longer wavelengths of light can penetrate through tissue to greater extent than shorter wavelengths. Longer wavelength fluorescence originating from deeper levels of tissue can decrease the contrast of the images acquired from more proximal layers of interest, thus degrading the image. The fourth problem was that the design goal of the older filters was to have less than 1% transmission at the crossover wavelengths between the lower boundary of the excitation filter and the upper boundary of the blocking filter. This still provided for the opportunity to have a significant cross-talk.
Thus, in view of the disadvantages and limitations of the current approaches, there remains a need for better methods and apparatus for autofluorescence imaging in the eye.
The state of the art is summarized in the references shown in Table I.
TABLE IREFERENCES1Delori FC, Dorey CK, Staurenghi G, et al. In vivo fluorescence of the ocularfundus exhibits retinal pigment epithelium lipofuscin characteristics. InvestOphthalmol Vis Sci 1995; 36: 718-2von Ruckmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescencewith a scanning laser ophthalmoscope. Br J Ophthalmol 1995;; 79: 407-12.3Eldred GE, Katz ML. Fluorophores of the human retinal pigment epithelium:separation and spectral characterization. Exp Eye Res 1988; 47: 71-86.4Eldred GE. Lipofuscin fluorophore inhibits lysosomal protein degradation andmay cause early stages of macular degeneration. Gerontology 1995; 41 (Suppl 2): 15-28.5Gaillard ER, Atherton SJ, Eldred G, Dillon J. Photophysical studies on humanretinal lipofuscin. Photochem Photobiol 1995; 61: 448-53.6Li W, Yanoff M, Li Y, He Z. Artificial senescence of bovine retinal pigmentepithelial cells induced by near-ultraviolet in vitro. Mech Ageing Dev 199922; 110: 137-557Yin D. Biochemical basis of lipofuscin, ceroid, and age-pigment-likefluoreophores. Free Radic Biol Med 1996; 21: 871-888.8.Reinboth JJ, Gautschi K, Munz K, Eldred GE, Reme CE. Lipofuscin in the retina:quantitative assay for an unprecedented autofluorescent compound (pyridiniumbis-retinoid, A2-E) of ocular age pigment. Exp Eye Res. 1997; 65: 639-43.9Suter M, Reme C, Grimm C, et al. Age-related macular degeneration. Thelipofuscin component n-retinyl-n-retinylidene ethanolamine detaches proapoptoticproteins from mitochondria and induces apoptosis in mammalian retinal pigmentepithelial cells. J Biol Chem 2000 15; 275: 39625-30.10Sparrow JR, Nakanishi K, Parish CA. The lipofuscin fluorophore A2E mediatesblue light-induced damage to retinal pigmented epithelial cells. Invest OphthalmolVis Sci. 2000; 41: 1981-9.11Liu J, Itagaki Y, Ben-Shabat S, Nakanishi K, Sparrow JR. The biosynthesis ofA2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane. J Biol Chem. 200022; 275: 29354-60.12Fishkin N, Jang YP, Itagaki Y, et al. A2-rhodopsin: a new fluorophore isolatedfrom photoreceptor outer segments. Org Biomol Chem. 2003 7; 1: 1101-5.13Spaide RF, Klancnik Jr JM. Fundus autofluorescence and central serouschorioretinopathy. Ophthalmology 2005; 112: 825-833.14Spaide, RF, Noble K, Morgan A, Freund KB. Vitelliform macular dystrophy.Ophthalmology 2006; 113: 1392-400.15Wing GL, Blanchard GC, Weiter JJ. The topography and age relationship oflipofuscin concentration in the retinal pigment epithelium. Invest Ophthalmol VisSci 1978; 17: 601-716von Ruckmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescencewith a scanning laser ophthalmoscope. Br J Ophthalmol 1995; 79: 407-1217Delori FC, Fleckner MR, Goger DG, et al. Autofluorescence distributionassociated with drusen in age-related macular degeneration. Invest Ophthalmol VisSci. 2000; 41: 496-504.18Spaide RF. Fundus autofluorescence and age-related macular degeneration.Ophthalmology 2003; 110: 392-9.